Molecular Genetic Studies in Arabidopsis thaliana: I. Cloning and Characterization of the Alcohol Dehydrogenase Gene; II. Complementation of an Alcohol Dehydrogenase Mutant; III. A Restriction Fragment Length Polymorphism Map of the Genome

Year: 1988 DOI: 10.7907/H8DS-A792
This thesis examines several genetic aspects of the flowering plant Arabidopsis thaliana which has previously been shown to possess several attributes for molecular genetic experiments such as a short life cycle, small size, fecundity, and a strong background of classical genetics. In particular, the nuclear genome is remarkably small and consists almost entirely of single copy sequences.

Two approaches are established for the isolation and study of plant genes using Arabidopsis as a model system. One approach demonstrates complementation of a mutant phenotype using the Arabidopsis alcohol dehydrogenase (ADH) gene. The ADH gene was first isolated from a genomic DNA library by cross-hybridization with a maize ADH1 gene probe. The gene structure was studied by DNA sequence analysis and mapping of the transcript. The gene conferred wild-type ADH activity to an Arabidopsis ADH null mutant when introduced by Agrobacterium-mediated transformation. Transformed plants were subjected to genetic and molecular analyses. The ADH gene may have utility as a gene tag in transformation experiments.

The second topic of this thesis is the construction of a genetic linkage map of restriction fragment length polymorphisms (RFLPs). The map provides an approach to cloning genes about which nothing more is known than a mutant phenotype and a map location because the RFLP markers can serve as starting points for the isolation of overlapping clones from a genomic DNA library. The map contains 90 RFLP markers distributed randomly throughout the nuclear genome. Since the genome consists of about 70,000 kilobase pairs, the markers are at an average physical spacing of approximately 780 kilobase pairs. The map is based on meiotic segregation of RFLPs in two different crosses detected with the restriction enzymes EcoRI, BglII, and XbaI. The RFLP linkage groups have been aligned with the standard genetic map of approximately 80 mutation markers.